Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

Abstract

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

Figure 1

Substrates and cofactors of dihydrofolate reductase (DHFR) [dihydrofolate (DHF) and nicotinamide adenine dinucleotide phosphate (NADP), respectively] and thymidylate synthase (TS) [5′-deoxyuridine monophosphate (dUMP) and N ⁵,N ¹⁰-methylene-5,6,7,8-tetrahydrofolate (mTHF), respectively]. Also shown are the two folate analogs and TS inhibitors raltitrexed (RTX) and pemetrexed (PTX).

Figure 2

Crystal structures of bifunctional homodimeric dihydrofolate reductase-thymidylate synthase from B. bovis (BbDHFR-TS). The DHFR subunit of each protomer (green, pink) is connected to a C-terminal TS subunit (violet, yellow) by a 40-­residue linker (cyan, gray). The protein in the apo state (top; PDB entry 3i3r) has a single chlorine ion in each TS active site (green spheres). Below are structures of BbDHFR-TS bound to dUMP, NADP and pemetrexed (middle; PDB entry 3k2h) and complexed with dUMP, NADP and raltitrexed (bottom; PDB entry 3nrr). Identical ligands are bound to protomer A (white, left) and protomer B (black, right) in each homodimer complex. Electron density (green mesh) is depicted for protomer A ligands at a 2.5σ contour level carved from a 1.6 Å atomic radius of the |F _o| − |F _c| maps using phases calculated from models lacking the ligand. This figure was created using CCP4 (Winn et al., 2011 ▶) and PyMOL (DeLano, 2002 ▶).

Figure 3

Key subunit interfaces for structures of BbDHFR-TS. Top left, arginine side chains from the opposite TS subunit (yellow) directly bind the terminal dUMP phosphate in the active site of one protomer (violet). Top right, the linker region from one protomer (gray) fits into a hydrophobic groove created by DHFR from the opposite protomer (green). Bottom, binding surface between the DHFR (pink), TS (yellow) and linker (gray) regions of protomer B of apo BbDHFR-TS, with labels for hydrogen-bonding residues. All distances are measured in Å. These figures were created using PDB entries 3i3r, 3k2h and 3nrr with PyMOL (DeLano, 2002 ▶).

Figure 4

Binding modes of pemetrexed (left) and raltitrexed (right) in the active sites of the TS (top) and DHFR (bottom) subunits of BbDHFRTS. Active sites from protomer A (TS, violet; DHFR, green; ligands, white) are overlaid with active sites from protomer B (TS, yellow; DHFR, pink; ligands, black) for both complexes. Raltitrexed (RTX) and pemetrexed (PTX) bind the TS active site identically in both protomers of both structures. For the DHFR subunit, the glutamate tail of PTX is consistent across both protomers of 3k2h, creating a salt bridge with Arg83. The glutamate tail of RTX is rotated relative to PTX in the DHFR subunit, resulting in loss of the Arg83 salt bridge, and binds in different conformations to each protomer of 3nrr. This figure was created using PyMOL (DeLano, 2002 ▶).